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Published 28 April 2003. doi:10.1083/jcb.200211095
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© The Rockefeller University Press, 0021-9525/2003/4/349 $5.00
The Journal of Cell Biology, Volume 161, Number 2, 349-358

Article

 Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification

Mimi Shirasu-Hiza1, Peg Coughlin2 and Tim Mitchison2

1 Department of Biochemistry, University of California, San Francisco, San Francisco, CA 94114
2 Department of Cell Biology, Harvard University Medical School, Boston, MA 02115

Address correspondence to Mimi Shirasu-Hiza, Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-3805. Fax: (617) 432-3702. E-mail: mshirasu{at}hms.harvard.edu

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth–promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.

Key Words: microtubule dynamics; microtubule-associated protein; XMAP215; GMPCPP; depolymerization

The online version of this article includes supplemental material.

* Abbreviations used in this paper: AS, ammonium sulfate; CPP MT, GMPCPP-stabilized MT; CSF, cytostatic factor; MT, microtubule.


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